Hollmann Emma K, Bailey Amanda K, Potharazu Archit V, Neely M Diana, Bowman Aaron B, Lippmann Ethan S
Department of Chemical and Biomolecular Engineering, Vanderbilt University, 2201 West End Ave, Nashville, TN, 37235, USA.
Department of Pediatrics, Vanderbilt University Medical Center, 1211 Medical Center Dr, Nashville, TN, 37232, USA.
Fluids Barriers CNS. 2017 Apr 13;14(1):9. doi: 10.1186/s12987-017-0059-0.
Due to their ability to limitlessly proliferate and specialize into almost any cell type, human induced pluripotent stem cells (iPSCs) offer an unprecedented opportunity to generate human brain microvascular endothelial cells (BMECs), which compose the blood-brain barrier (BBB), for research purposes. Unfortunately, the time, expense, and expertise required to differentiate iPSCs to purified BMECs precludes their widespread use. Here, we report the use of a defined medium that accelerates the differentiation of iPSCs to BMECs while achieving comparable performance to BMECs produced by established methods.
Induced pluripotent stem cells were seeded at defined densities and differentiated to BMECs using defined medium termed E6. Resultant purified BMEC phenotypes were assessed through trans-endothelial electrical resistance (TEER), fluorescein permeability, and P-glycoprotein and MRP family efflux transporter activity. Expression of endothelial markers and their signature tight junction proteins were confirmed using immunocytochemistry. The influence of co-culture with astrocytes and pericytes on purified BMECs was assessed via TEER measurements. The robustness of the differentiation method was confirmed across independent iPSC lines.
The use of E6 medium, coupled with updated culture methods, reduced the differentiation time of iPSCs to BMECs from thirteen to 8 days. E6-derived BMECs expressed GLUT-1, claudin-5, occludin, PECAM-1, and VE-cadherin and consistently achieved TEER values exceeding 2500 Ω × cm across multiple iPSC lines, with a maximum TEER value of 4678 ± 49 Ω × cm and fluorescein permeability below 1.95 × 10 cm/s. E6-derived BMECs maintained TEER above 1000 Ω × cm for a minimum of 8 days and showed no statistical difference in efflux transporter activity compared to BMECs differentiated by conventional means. The method was also found to support long-term stability of BMECs harboring biallelic PARK2 mutations associated with Parkinson's Disease. Finally, BMECs differentiated using E6 medium responded to inductive cues from astrocytes and pericytes and achieved a maximum TEER value of 6635 ± 315 Ω × cm, which to our knowledge is the highest reported in vitro TEER value to date.
Given the accelerated differentiation, equivalent performance, and reduced cost to produce BMECs, our updated methods should make iPSC-derived in vitro BBB models more accessible for a wide variety of applications.
由于人类诱导多能干细胞（iPSC）能够无限增殖并分化为几乎任何细胞类型，这为生成构成血脑屏障（BBB）的人脑微血管内皮细胞（BMEC）用于研究目的提供了前所未有的机会。不幸的是，将iPSC分化为纯化的BMEC所需的时间、费用和专业知识阻碍了它们的广泛应用。在此，我们报告使用一种特定培养基，该培养基可加速iPSC向BMEC的分化，同时实现与传统方法产生的BMEC相当的性能。
将诱导多能干细胞以特定密度接种，并使用称为E6的特定培养基将其分化为BMEC。通过跨内皮电阻（TEER）、荧光素通透性以及P-糖蛋白和多药耐药相关蛋白（MRP）家族外排转运蛋白活性来评估所得纯化BMEC的表型。使用免疫细胞化学法确认内皮标志物及其标志性紧密连接蛋白的表达。通过TEER测量评估与星形胶质细胞和周细胞共培养对纯化BMEC的影响。在独立的iPSC系中证实了分化方法的稳健性。
使用E6培养基并结合更新的培养方法，将iPSC向BMEC的分化时间从13天缩短至8天。E6来源的BMEC表达葡萄糖转运蛋白1（GLUT-1）、闭合蛋白-5（claudin-5）、闭锁蛋白（occludin）、血小板内皮细胞黏附分子-1（PECAM-1）和血管内皮钙黏蛋白（VE-cadherin），并且在多个iPSC系中始终实现超过2500 Ω×cm的TEER值，最大TEER值为4678±49 Ω×cm，荧光素通透性低于1.95×10 cm/s。E6来源的BMEC将TEER维持在1000 Ω×cm以上至少持续8天，并且与通过传统方法分化的BMEC相比，其外排转运蛋白活性无统计学差异。还发现该方法支持携带与帕金森病相关的双等位基因PARK2突变的BMEC的长期稳定性。最后，使用E6培养基分化的BMEC对来自星形胶质细胞和周细胞的诱导信号有反应，并且实现了最大TEER值为6635±315 Ω×cm，据我们所知，这是迄今为止报道的最高体外TEER值。
鉴于分化加速、性能相当且生产BMEC的成本降低，我们更新的方法应使iPSC来源的体外血脑屏障模型更易于广泛应用。
